Saturday, November 23, 2024
11/23/2024
SUMMARY My lab has pioneered studies defining how cells initiate and maintain a quiescent state in the face of nutrient deprivation and quantifying the role of copy number variation in rapid adaptive evolution. In this proposal, we have outlined two central areas of research that extend this work. The first project is aimed at investigating how protein expression programs and cellular biology is remodeled in quiescent cells. Cell quiescence is the dominant state of all cells, but the most poorly understood. Our prior research has defined the genetic requirements for quiescence and quantified genetic interactions with evolutionarily conserved signaling pathways that are required for quiescence. In our proposed research we will study the dynamics of gene expression remodeling in quiescent cells at the level of protein expression using metabolic labeling and mass spectrometry. We will define the role of key signaling pathways by studying defects in gene expression remodeling in strains mutant for TORC1, PKA, AMPK, and PHO85. To study remodeling of the cellular environment in quiescence we will use genetically encoded multimeric nanoparticles (GEMs) and live cell imaging to quantify changes in cellular crowding in quiescent cells. We will also study mutants in vacuole biogenesis to test the role of this organelle in reorganizing the cellular environment in quiescence. We will define genetic factors that contribute to drug tolerance in quiescent cells and leverage this information to test approaches to increasing antifungal drug effectiveness in quiescent cells in the fungal pathogens Candida albicans and Candida glabrata. Our second project is aimed at understanding the dynamics of copy number variation in evolving populations and their functional consequences. Copy number variants (CNVs) are a prevalent source of genetic variation in humans, in which they underlie both heritable diseases and pathogenic somatic variation. CNVs underlie phenotypic variation in a range of organisms and often confer resistance to therapeutic treatment in pathogenic microbes. Our studies have demonstrated that CNVs are frequent drivers of rapid adaptive evolution during microbial experimental evolution in chemostat cultures. We have developed a combined CNV reporter and lineage tracking system that enables accurate quantification of CNV dynamics in efficient methods for multiplexed mutant analysis. Using a combination of these approaches, we will study the dynamics of CNV-mediated adaptive evolution in fluctuating environments. We will then test the fitness effects of CNVs in conditions in which they have been selected, in which we expect them to confer a fitness benefit, and a diversity of additional conditions, in which we expect them to be neutral or deleterious. By incorporating known features of CNVs, we will build a quantitative model of their fitness costs and benefits. To investigate the functional effects of CNVs we will quantify their gene expression consequences at the level of mRNA abundance and ribosome occupancy and test their effect on gene expression heterogeneity using single cell RNA sequencing.
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